The levels of squamous cell carcinoma antigen (SCC-Ag) and C-reactive protein (CRP) can be used to predict tumor invasion, lymph node metastasis, staging and survival in patients with oral cavity cancer. The present study analyzed the relationship between pre-treatment levels of SCC-Ag and CRP in relation to clinicopathological factors in patients with pharyngolaryngeal cancer (PLC) and determined whether elevated levels of CRP and SCC-Ag were associated with tumor metabolic activity via [18F] fluorodeoxyglucose positron emission tomography (FDG-PET). We retrospectively recruited one hundred and six PLC patients between June 2008 and December 2011. All patients received computed tomography (CT)/magnetic resonance imaging (MRI) and FDG-PET staging analyses, and the serum levels of SCC-Ag and CRP in these patients were measured prior to treatment. A SCC-Ag level ≥2.0 ng/ml and a CRP level ≥5.0 mg/L were significantly associated with clinical stage (P<0.001), clinical tumor status (P<0.001), and clinical nodal status (P<0.001). The elevation of both SCC-Ag and CRP levels was correlated with the standardized uptake value (SUV) max of the tumor (≥8.6 mg/L) and lymph nodes (≥5.7 ng/ml) (P = 0.019). The present study demonstrated that the presence of high levels of both pre-treatment SCC-Ag and CRP acts as a predictor of clinical stage, clinical tumor status, and clinical nodal status in patients with PLC. Moreover, elevated levels of SCC-Ag and CRP were associated with a high metabolic rate as well as the proliferative activity measured according to the SUVmax of the tumor and lymph nodes. Therefore, elevated levels of these two factors have the potential to serve as biomarkers for the prediction of tumor aggressiveness in cases of PLC. Head and neck cancer is the fourth most common cancer and leading cause of cancer-related deaths in Taiwan. Knowledge of prognostic factors would be beneficial when evaluating and counseling patients with these cancers. Notably, HPV infection status has been strongly associated with the therapeutic response and survival of oropharyngeal cancer patients; however, it was not shown to be related to tumor stage or clinicopathological factors. in vitro postoperatively, including mitotic count and the Ki-67 labeling index. Fluorodeoxyglucose positron emission tomography (FDG-PET) is a well-established tool for evaluating head and neck cancer. In this study, we investigated the significance of SCC-Ag and CRP levels in PLC patients and their relationship with various clinicopathological factors as well as 18F-FDG uptake levels on PET scans.We retrospectively reviewed the charts of all patients newly diagnosed with PLC at our institute between June 2008 and Aug 2011. Patients with distant metastases at diagnosis or who were lost to follow up following diagnosis were excluded. Follow-up commenced at the time of cancer diagnosis and, for this study, completed at the earlier of either December 2011 or death. All patients were evaluated preoperatively with history, examination, routine bloods, chest radiograph, liver ultrasound, FDG-PET and either CT or magnetic resonance imaging (MRI) of the head and neck. 2 cisplatin (P) on Day 1 followed by 800 mg/m2 oral tegafur (T) per day and 60 mg oral leucovorin (L) per day for 14 days (PTL regimen). All patients were staged as per American Joint Committee on Cancer guidelines , lymph node extra-capsular spread (ECS), tumor depth ≥10 mm or poor tumor differentiation received postoperative radiotherapy or CCRT 4–8 weeks after surgery. Radiotherapy involved a three-field technique and consisted of conventional bilateral opposing fields with a matching anterior lower neck portal. Daily fractionation size was 1.8 or 2 Gy and five fractions were delivered per week. The planning target volume was created by adding a 5 to 7 mm margin to the clinical target volume. For the group receiving radical surgery first, the post-operative radiotherapy dose was 60–68.4 Gy, depending on the pathology risk factor; for the organ preservation group, the dose range was 68.4–76 Gy.Pre-treatment serum levels of CRP and SCC-Ag were measured in a fresh blood sample obtained at the time of diagnosis, prior to any form of medical intervention, to minimize inter-individual differences. Serum CRP levels were detected with a high-sensitivity assay  using an auto-analyzer . Serum SCC-Ag level was measured using a commercially available chemiluminescent microparticle immunoassay . Serum CRP cutoff was set at 5.0 mg/L as this level is internationally agreed to indicate inflammation. 18F-FDG administered intravenously. An oral contrast agent was administered during uptake time. Next, PET/CT scans  combining a 16-slice spiral CT scanner were performed. The patients were scanned from head to mid-thigh; lower limbs were scanned if indicated. A positive finding was defined as a focus of increased 18F-FDG uptake with intensity higher than that of the surrounding tissues, which was localized according to hybrid images in an area that did not correspond to the physiologic bio-distribution of the radiotracer. Regions of interest (ROIs) were measured for lesions visible on PET images, as well as on simultaneously displayed axial, coronal, and sagittal tomograms. FDG-PET/CT was used for the initial tumor survey. Patients fasted for 6 hours before scans and were then subjected to 370 to 444 MBq (10 to 12 mCi), with rd, 6th and 12th month for the first year and then annually for the following 4 years. Patients who presented with abnormal clinical symptoms/signs or laboratory data underwent a bone scan and liver ultrasound for further evaluation.Patients were followed monthly during the first 6 months after treatment, every 2 months for the following 6 months, every 3 months during the second year, and every 6 months thereafter. The patients were subjected postoperatively to hemograms, blood chemistry measurements, chest x-rays, and CT or MRI analyses at the 3The study was approved by the Institutional Review Board of Chang Gung Memorial Hospital, Linkou (IRB). The data (CRP and SCC-Ag) were analyzed in retrospective manner. The information was recorded by the investigator in a manner that subjects could not be identified directly or through identifiers linked to the subjects. No informed consent was requested by the IRB.The following major variables were examined: CRP levels, SCC-Ag levels, SUV max of the primary tumor and neck lymph nodes, and clinical and pathological lymph node status. The statistical methods used included a univariate analysis with the chi-squared test, and a univariate analysis of survival differences was performed using the log-rank test. A two-sided P value <0.05 was considered statistically significant. These analyses were performed using the Statistical Package for the Social Sciences (SPSS) statistical software, version 19.0 .Between June 2008 and August 2011, 106 consecutive patients  were newly diagnosed with PLC at our institute. The sites of their primary tumors, their clinical staging and the treatments they received are detailed in 2 trend test: P = 0.005), clinical nodal status (χ2 trend test: P = 0.003), and tumor differentiation (χ2trend test: P = 0.014) (2 trend test: P = 0.001), clinical tumor status (χ2 trend test: P<0.001), and clinical nodal status (χ2 trend test: P = 0.001). Furthermore, higher SCC-Ag levels were often accompanied by higher serum CRP levels (χ2 test: P = 0.006).The cutoff point for measurements of serum CRP level was set at 5.0 mg/L. A close association was observed between a higher CRP level (CRP≥5.0 mg/L) and clinical stage (χ= 0.014) . A close2 trend test: P<0.001), clinical tumor status (χ2 trend test: P<0.001), and clinical nodal status (χ2 trend test: P<0.001)  and CRP (≥5.0 mg/L) and clinical stage (χP<0.001) .We next compared the association between CRP level, SCC-Ag level, SUVtumor-max and SUVnodal-max and found that the levels of CRP and SCC-Ag were not associated with SUVtumor-max . However, a significant association between CRP level, SCC-Ag level and SUVnodal-max  was observed.2 trend test: P<0.001), clinical tumor status (χ2 trend test: P<0.001), and nodal status (χ2 trend test: P<0.001). The associations between the 4 patient groups according to SUVtumor-max and SUVnodal-max and according to the pre-treatment levels of SCC-Ag and CRP were also analyzed and found to be significant (P = 0.019)  and SUVnodal-max (≥5.7) and clinical stage  and CRP (P = 0.010) had significant influences on overall survival (OS) in the univariate analysis. We combined the CRP and SCC-Ag levels as a variable value for the analysis of prognosis, for which the patients were divided into four groups. For the analysis of all 103 patients in the study, the OS with higher SCC-Ag and higher CRP levels (n = 27) was significantly different to that with non-elevated levels of SCC-Ag and CRP (n = 76) , although the disease-free survival (DFS) of patients was not significantly different between groups .The squamous cell carcinoma antigen (SCC-Ag) is a tumor-associated protein that was first isolated from SCC tissues of the uterine cervix. Serum CRP level is a sensitive marker of inflammation that is elevated in response to tissue damage or infection and has been shown to be a prognostic factor in OSCC. Reports from the study of oral cavity SCC have suggested a relationship between CRP level and poor outcomes, pathological tumor status, nodal status and lymph node ECS. FDG-PET has been increasingly applied in head and neck cancer patients for staging and for the study of tumor aggressiveness through the measurement of SUV. The combined measurement of SCC-Ag and CRP revealed a more significant correlation with clinical status than either SCC-Ag or CRP when used alone. The patient subgroup with positive levels of both SCC-Ag and CRP demonstrated a profoundly significant association with clinical stage (P<0.001), clinical tumor status (P<0.001) and nodal status (P<0.001) compared to other patient groups . The comWhen tumor cells proliferate rapidly, there is an increase in glucose consumption, which be observed as an increase in glucose uptake on a FDG-PET scan. The destruction of nearby tissue and lymph node metastasis is generally accompanied by tissue inflammation, and the infiltration of inflammatory cells and the production of cytokines, especially IL-6, increase the CRP level in the serum. In addition, T-lymphocytes located at the periphery of tumors serve to increase the production of SCC-Ag, We further analyzed the significance of SCC-Ag and CRP in PLC and found no significant correlation between SCC-Ag level, DFS and OS in a univariate analysis  . In addiThe present study demonstrated that the combined measurement of SCC-Ag and CRP levels served as a marker of clinical status in PLC and may represent a biomarker capable of predicting prognosis. However, further work is required to elucidate the precise molecular mechanisms for this observed interaction between SCC-Ag and CRP in PLC. As measurements of the levels of SCC-Ag and CRP can be performed quickly, inexpensively and repeatably in a clinical setting, we believe that the levels of SCC-Ag and CRP, as well as the combination of these two factors, could serve as relevant biomarkers of tumor aggressiveness prior to treatment in patients with PLC.Table S1Literature discussing the role of C-reactive protein (CRP) in oropharyngeal cancer.(DOCX)Click here for additional data file.


After the F2 weight gained 20-25 g, their tendency for right-handedness or Left-handedness and response to the new environment was determined by T-maze and open field method, respectively. In addition, the effects of morphine, sulpiride and dextromethorphan on the animals’ motor activity were studied.Twenty four pregnant NMRI female mice (W: 25 g) were divided into the experimental and control groups (n = 12/group). Animals in the experimental group were kept in the restraint cylinder  for 60 min/day for 15 consecutive days, while the control group did not receive stress. On the 8Results showed that plasma corticosterone level in the experimental group was elevated significantly with respect to the controls. In the off-springs, left-handedness was more frequent in both the male and female animals whose mothers experienced restrained stress. In the open field paradigm, however, the females of experimental group showed more activity compared to the others. While the females of the control group showed more response to morphine (50 mg/Kg), interestingly, both male and female animals in the experimental group showed hypo activity to morphine . Similarly, sulpiride (25 and 50 mg/Kg) reduced the animals’ activity in both groups, while dextromethorphan did not cause any difference. In conclusion, it can be summarized that stress during the gestation period may change the response to the morphine-induced motor activity, in a sex-dependent manner. Stress is considered as an important factor which can influence embryos growth during the gestation period of life. Stress has been defined as any condition that changes internal or external melio. Stress reaction differs in accordance with its severity to threats in life , 2. In gAnimals×15×20 cm) over the night for mating. At 8:0.0 PM the following day, the animals were separated and the Embryonic Zero day (E0) was detected through observing the vaginal plug and/or identifying the sperm in vaginal smear. The pregnant mice were randomly divided into control and experimental groups (n = 12/group). The animals were housed 2/cage and remained in a standard animal room conditions (22 ± 2°C and 50% humidity with 12 h light/dark cycle) and have free access to standard mouse chow and tap water add lib except during the experiments.Thirty-two male and female NMRI mice  were housed one by one in a mating cage All experiments took place in light time (10:00-12:00). To induct the stress, each animal was placed in a PVC cylinder  in which one of the ends was blocked , 14 and Blood sampling and plasma corticosterone levels detection in stressed pregnant miceBlood samples were taken from retro-orbital sinus (0.1 mL of the blood in 0.9 mL sodium citrate 5%) of the pregnant mice on Embryonic day 8 (E8) of pregnancy between 10:00-12:00 , 14. TheDetermination of left-handedness and right-handedness in F2 generation When the off-springs’  weight reached up to 20 g, the animals were placed in a T-maze apparatus and their tendency for left or right arm of the maze was estimated . All experiments were taken place in a silent room from 10:0.0 AM to 17:0.0 PM. The experiment was repeated three times for each animal and the animal’s arm which was selected more than once , was considered as its tendency. The experiments took place in separate rooms for males and females.Locomotion activity in F2 generatione.g. F2) were engaged in locomotor activity experiments. All experiments took place in a silent room from 10:0.0 AM till 17:0.0 PM  were brought to the lab and remain there 1 h for adaptation. These animals were different from the animals used in the left-handedness and right-handedness determination. Each animal was placed in an open field apparatus (a cylinder with 30 cm diameter and 30 cm high made by Plexiglas), and after 5 min, its locomotion activity was recorded for 10 min by a digital camera. These experiments also took place in separate rooms for male and female mice.After right-handedness and left-handedness determination, the next generations , ± sulpiride ([±]-N-1-[Ethylpyrrolidin-2-ylmethyl])-2-methoxy-5-sulfamoylbenzamide),  and dextromethorphan hydrobromide  were dissolved in normal saline (10 mL) and were injected in volume of 0.1 mL/10 g per body weight. Morphine was injected subcutaneously (s.c.). Sulpiride and dextromethorphan were injected intra-peritoneally (IP) 30 min before the beginning of experiments. The doses for all drugs were chosen according to the previous studies -17.Statistical analysisData are shown as mean ± SEM. One-way analysis of variance (ANOVA) followed by Tukey post-test, Un-Paired t-test or chi-square tests was applied. p < 0.05 considered statistically significant.Effects of stress on plasma corticosterone level in pregnant miceThe effect of stress on blood corticosterone level in pregnant animals is shown in Determination of right or left-handedness in off-springsWhen the off-springs of the control group were placed in T-maze, more than 80% of the males and females preferred the right arm . However, there sults of off-springs in experimental mothers were completely reversed as up to 70% of both males and females preferred their left arm to the right one  induced hypoactivity in all animals (N- methyl-D- aspartate (NMDA) glutamate receptor antagonist, dextromethorphan,  did not change the animals’ locomotor activity in all groups.By injecting different doses of dopamine Di.e. restraint in a closed tube) can increase plasma corticosterone level in the pregnant mice. Second, the study revealed that off-springs from stressed mothers had different tendency for left or right preference, and third, off-spring’s responsibility to morphine was different according to their sex.First of all, we found that psychological stress  also showed difference in the experimental group. While the controls showed no response to low doses of morphine, the response of the experimental group was even a decline. In agreement with our results, Michaels et al. showed that the early post-natal stress in rats can change the response to the mu-opioid receptor agonists in place conditioning paradigm  receptor antagonist (In the last part of our experiments, dextromethorphan as tagonist  was admi2 generation brain laterality was changed in animals whose mothers experienced milled stress. Second, response to morphine-induced locomotion was also altered in the experimental group which was sex dependent but could not be linked to D2 dopamine and NMDA glutamate receptor dysfunction.In conclusion, results indicated that first, F


Axonal transport plays a crucial role in neuronal morphogenesis, survival and function. Despite its importance, however, the molecular mechanisms of axonal transport remain mostly unknown because a simple and quantitative assay system for monitoring this cellular process has been lacking. In order to better characterize the mechanisms involved in axonal transport, we formulate a novel computer-assisted monitoring system of axonal transport. Potential uses of this system and implications for future studies will be discussed. Neurons are highly polarized cells having axons and dendrites, with proper neuronal differentiation, survival and function heavily dependent on the axonal and dendritic transport machinery ,2. In axFor the analysis of axonal transport, conventional assay systems have been utilized, such as isotope tracer techniques, video-enhanced differential interference contrast microscopy or fluorescence recovery after photobleaching (FRAP) via the introduction of green fluorescence protein (GFP)-tagged proteins . CurrentIn the peripheral and central neurons, neurotrophins are important regulators for the survival, differentiation and maintenance of nerve cells. Recent studies indicate that axonal transport plays an important role in neuronal survival ,14. In fDuring development, after postmitotic division, neurons extend their neurites, and recognize their environments to make synapses with their appropriate target cells. Although a role of local protein synthesis in growth cone has now been appreciated in this process, all the proteins required in the axon, growth cone and synaptic terminal must be transported down the axon after they are synthesized in the cell body. In neurons, various kinds of receptors for axon guidance molecules must be localized properly to axons or dendrites for appropriate neuronal wiring. Signaling outcomes downstream of ligand binding are determined by the location, levels and residence times of receptors on the neuronal plasma membrane. Therefore, the mechanisms controlling the trafficking of these receptors are crucial to the proper wiring of circuits. Indeed, the molecular machinery involving membrane trafficking has been implicated in axon guidance.unc-51 Caenorbabditis elegans mutants, some axon guidance defects have been observed . We . We 4]. a3A-EGFP . An EGFPa3A-EGFP .Various kinds of therapeutic agents have neurological side effects, and some have been associated with the impairment of axonal transport ,27. Howe50 values for antero- or retrograde axonal transport of paclitaxel, vincristine and cisplatin were 4.1 or 5.5 nM, 100 or 130 pM, and 3.0 or 5.5 μM, respectively. These values are comparable to the effective concentrations that inhibit the growth of tumor cells [Many anti-cancer drugs have neuropathic side effects. For example, paclitaxel stimulates and vincristine inhibits the polymerization of β-tubulin to α-tubulin, resulting in the appearance of microtubule bundles and some aberrant structures in the mitotic phase of the cell cycle . A possior cells –40, furtUsing this system, we have discovered that the effects of these pharmacological agents have differential effects on each component of axonal transport. Oxaliplatin shows a tendency to inhibit the fast component of antero- and retrograde axonal transport with a velocity faster than 2 μm/s . On the 2+- and Mg2+-free phosphate buffered saline at 37 °C for 6 min, and stopped by adding trypsin-free medium, as described [l-lysine-coated 35-mm glass base dish . The cells were then cultured at 37 °C for 12–14 h. The cultured cells were washed three times in Leibovitz’s L-15 medium (L-15 medium) , and cultured for 30 min in L-15 medium containing CM-DiI (2 mM) and 2.5 S nerve growth factor . The cells were washed three times with L-15 medium, then incubated for 6 h in the CM-DiI-free culture medium to remove any excess dye loaded in the DRG neurons. The absolute value of the number of DiI-labeled moving particles (/2 min) at the start of the experiments varied among the DRG neurons examined. This was probably due to certain differences in the culture conditions and/or developmental stages of the neurons. To characterize the particles stained with CM-DiI, double staining with CM-DiI and mitoTracker or erTracker was performed in DRG neurons. The procedures involving the experimental animal complied with the animal care guidelines of the National Institutes of Health and the animal ethics committee of Yokohama City University.CM-DiI was purchased from Invitrogen . Poly-L-lysine, sodium azide, nocodazole, vinblastine sulfate, cisplatin, oxaliplatin, 5-fluorouracil, cytochalasin B and lidocaine were from Sigma . Paclitaxel was from Wako . Methotrexate was from Fluka . DRGs were removed from embryonic day-7 chick embryos, and were treated with 0.25% trypsin in Caescribed . The celCM-DiI-labeled moving particles in the axons of cultured DRG neurons were observed using a laser-scanning microscope with a water-immersed objective set at ×40 (C-Apochromat/1.2 W corr) equipped with an Axioplan 2 imaging microscope (Carl Zeiss). The images were typically 644 × 130 pixels, corresponding to an image size of 75 × 15.1 μm using a pixel size of 0.12 μm. The images were collected every 0.5 μs, at intervals of 0.25 μs, for 2 min.t-test and Mann-Whitney U-test.Data are expressed as means ± S.E.M. of n experiments. The results were analyzed using Student’s In conclusion, we have established a system which allows prompt and accurate analysis of axonal transport that can visualize moving particles, and which may be applied together with other probes and recent imaging techniques. Future studies may include application of this assay system to analyze frequency of movement measured, distance traveled and other complex patterns of movement per cargo particle. This system affords a chemical biology strategy for evaluating neurotoxic effects of candidate therapeutic agents and chemicals for side-effect, hazard and risk assessment, and for developing novel therapeutic agents to restore affected axonal transport in various kinds of neuronal cells originating from central and peripheral nervous tissue, or disease-related pluripotent stem cells .


The platform chemical lactate is currently produced mainly through the fermentation of sugars presented in biomass. Besides the synthesis of biodegradable polylactate, lactate is also viewed as a feedstock for the green chemistry of the future. Pyruvate, another important platform chemical, can be produced from lactate through biocatalysis.Pseudomonas stutzeri SDM catalyze lactate oxidation with lactate-induced NAD-independent lactate dehydrogenases (iLDHs) through the inherent electron transfer chain. Unlike the lactate oxidation processes observed in previous reports, the mechanism underlying lactate oxidation described in the present study excluded the costliness of the cofactor regeneration step and production of the byproduct hydrogen peroxide.It was established that whole cells of dl-lactate as the substrate and whole cells of the lactate-utilizing P. stutzeri SDM as catalyst. Under optimal conditions, the biocatalytic process produced pyruvate at a high concentration (48.4 g l−1) and a high yield (98%). The bioconversion system provides a promising alternative for the green production of pyruvate.Biocatalysis conditions were optimized by using the cheap  Lactic acid, the most important hydroxycarboxylic acid, is currently commercially produced by the fermentation of sugars present in biomass 2 rather than pyruvate Most of the reported chemical catalysts convert a major part of lactate to acetaldehyde and COPseudomonas stutzeri SDM was reported to have the ability to produce pyruvate from lactate with oxygen as the terminal electron acceptor P. stutzeri SDM catalyzed lactate oxidation, which made the strain a promising biocatalyst for the commercial pyruvate production. NAD-independent lactate dehydrogenases (iLDHs) were reported to be involved in the lactate oxidation process dl-lactate, by the lactate-utilizing strain SDM were also developed.In a previous report, P. stutzeri SDM possesses 2 inducible iLDHs that made the strain a good biocatalyst for the 2-oxo-carboxylate production 3, and salicylhydroxamic acid on the pyruvate-producing activity of the P. stutzeri SDM crude extract were studied. As shown in c reductase inhibitor antimycin A distinctly inhibited the pyruvate-producing activity. This implied that iLDHs in P. stutzeri SDM might use quinone as the natural electron acceptor. The electron acquired by quinone would be transferred to cytochrome (by cytochrome c reductase) and then terminally transferred to oxygen (by cytochrome oxidase). In addition to the traditionally cytochrome oxidase, Pseudonomas strains also have some alternative cytochrome oxidase, such as cytochrome cbb3 oxidase 3, and salicylhydroxamic acid) exhibited different inhibition effects on the l- and d-lactate oxidation activities of P. stutzeri SDM due to the complexity of the cytochrome oxidase system in Pseudonomas strains, those results implied the roles of cytochrome in the lactate oxidation process.c fraction was purified from P. stutzeri SDM  concentrations, the effect of DO on the biotransformation activity of P. stutzeri SDM was studied in a 5-l reactor. As shown in In the case of the lactate oxidation mechanism in d-Lactate or l-lactate has been employed in pyruvate production dl-lactate was used as the substrate for pyruvate production in this work.−1 of cell biomass of P. stutzeri SDM and 0.4 M dl-lactate at 30°C. As shown in Influence of reaction pH on pyruvate production was determined in 100 mM phosphate buffer containing 6 g dry cell weight (DCW) ldl-lactate on biocatalysis was investigated to determine its optimal range. The dl-lactate concentration was varied from 0.15 to 0.75 M. As shown in dl-lactate was 0.45 M. High DL-lactate concentration (more than 0.6 M) would lead to substrate inhibition and resulted in lower pyruvate concentrations.Substrate concentration was also important for the conversion of DL-lactate to pyruvate. Effect of the concentration of To investigate the influence of reaction temperature on pyruvate production, the reaction was carried out at different temperatures at pH 8.0. As shown in –1 of P. stutzeri SDM as the biocatalyst. As shown in –1 of P. stutzeri SDM was optimal and produced the highest pyruvate concentration and relatively high specific biotransformation activity.Biocatalysis was carried out with 2–16 g DCW ll-lactate The biocatalysis production of pyruvate from lactate was a bio-oxidation process. Oxygen would be the terminal electron acceptor. Thus, oxygen was also the substrate of the biocatalysis process and the effect of DO on pyruvate production should be investigated. As shown in dl-lactate was developed. The biocatalysis was conducted at 30°C in distilled water (pH was adjusted to 8.0) with 10 g DCW l−1 of P. stutzeri SDM as the biocatalyst. The DO saturation was controlled at 15%. The dl-lactate concentration in the 5-l reactor was about 0.45 M. As shown in dl-lactate after 29 h of biotransformation.Combining these results, an optimal biotransformation system for the production of pyruvate from −1) and high volumetric productivity (6 g l−1 h−1) of pyruvate have been obtained through fermentation Pyruvate is an important starting material widely applied in chemical, pharmaceutical, and agrochemical industries l-lactate with oxygen as the second substrate, giving hydrogen peroxide as a byproduct. Hydrogen peroxide decomposes pyruvate to acetate, carbon dioxide, and water, lowering the production yield. If the problem of further oxidation of pyruvate by hydrogen peroxide can be solved, the biocatalysis production of pyruvate from lactate has the potential to be commercialized because of the low price of lactate P. stutzeri SDM was confirmed to have the ability to produce pyruvate from lactate with the merit of no hydrogen peroxide production. iLDHs in P. stutzeri SDM firstly acquired the electron from the lactate and then the electron would be terminally transferred to oxygen. However, the electron transfer process has never been clarified in previous works.Lactate, which can be easily produced from biomass, is the most promising substrate in the biocatalysis production of pyruvate R)-hydroxycarboxylate-viologen-oxidoreductase (HVOR) in Proteus vulgaris or Proteus mirabilis with the addition of artificial redox mediators was studied in previous reports P. stutzeri SDM, pyruvate was produced without the addition of an artificial redox mediator. The reduction of cytochrome c in P. stutzeri SDM by dl-lactate implied that the electron transfer components of respiratory chain might play an important role in lactate oxidation. Effects of different respiratory chain inhibitors on the pyruvate production further identified the involvement of the electron transfer chain in the lactate oxidation process. Unlike Proteus strains, the cofactor regeneration of iLDHs in P. stutzeri SDM may employ the inherent electron transfer system of the strain. This process, which excludes the expensive cofactor regeneration step, makes P. stutzeri SDM a rather practicable alternative for the biocatalytic production of pyruvate. Under the optimal conditions, with biocatalyst prepared from 10 g DCW l−1 of P. stutzeri SDM, 0.44 M pyruvate was obtained after 29 h.Pyruvate production by  and a high yield (98%). The biocatalysis process introduced in this study provides not only a rather feasible method for pyruvate production but also a green pathway for the utilization of lactate produced from biomass.In conclusion, the lactate-utilizing l-Lactate and bovine serum albumin were purchased from Sigma. d-Lactate was purchased from Fluka. dl-Lactate was purchased from Wujiang Ciyun Flavor & Fragrance Co. Ltd. (P. R. China). Diphenylamine, antimycin A, and salicylhydroxamic acid were purchased from Sigma. All other chemicals were of reagent grade.P. stutzeri SDM (China Center for Type Culture Collection No. M 206010) isolated from soil was used −1dl-lactate was used as the fermentation medium dl-lactate as the sole carbon source were resuspended in 50 mM Tris-HCl (pH 8.0) and disrupted by sonication  in an ice bath. The disrupted cells were subjected to centrifugation for 20 min at 12,000 g, and the supernatant was used as crude cell extract.Cells of the SDM strain grown in MSM containing P. stutzeri SDM was prepared from the crude cell extract by centrifugation at 147,000 g for 180 min. Triton X-100  was added to the membrane fraction to a final concentration of 1 mg mg−1 of protein. The supernatant (detergent extract) was applied to a column of DEAE Sepharose Fast Flow equilibrated with Buffer A: 50 mM Tris-HCl (pH 8.0) containing 1 mM dithiothreitol (DTT), 5 mM MgSO4, 0.1% Triton X-100, and 1 mM EDTA. The column was washed with Buffer B: 50 mM Tris-HCl (pH 8.0) containing 1 mM DTT, 200 mM KCl, 5 mM MgSO4, 0.1% Triton X-100, and 1 mM EDTA at a flow rate of 5 ml min−1. Cytochrome from column effluents was monitored by measuring the absorbance at 410 nm. The fractions containing cytochrome were concentrated by ultrafiltration and desalted with gel G-25. The cytochrome pool after desalting was then applied to a column of DEAE A-25 pre-equilibrated with Buffer A. The column was washed with a linear gradient of 0–100% Buffer B at a flow rate of 0.5 ml min−1. The fractions containing cytochrome were concentrated and stored at 0 to 4°C. Native polyacrylamide gel electrophoresis (native-PAGE) was performed on a 5–12% native polyacrylamide gradient gel with a Mini-Protean III system (Bio-Rad) according to the protocol by Davis The membrane fraction of l-lactate or d-lactate. The reaction was initiated by the addition of whole cells or crude cell extract of SDM at 37°C for 10 min. The reaction was stopped by adding 0.05 ml of 1 M NaOH. The mixture was subjected to centrifugation for 1 min at 16,000 g, and a 0.5-ml portion of the supernatant was used for pyruvate detection. One unit was defined as the amount of enzyme that converted lactate to 1.0 µM pyruvate per minute under the test conditions.Pyruvate-producing activity was determined in 1 ml of 67 mM phosphate buffer (pH 7.4) containing 20 mM −1 with 10 mM H2SO4 as eluent at 55°C pro UV/visible spectrophotometer . Protein was determined by the Markwell variation of the Lowry method, with bovine serum albumin as the standard Accurate concentrations of lactate and pyruvate were analyzed by HPLC  using an Aminex HPX-87H column (Bio-Rad), which was running at 0.4 ml min


Because glutamate dissociates from subsets of NMDA receptors very slowly, excitatory synaptic transmission can leave a silent residual trace that outlasts the electrical activity by hundreds of milliseconds. Read-out of the memory trace is possible if a critical level of these bound-but-blocked receptors accumulates on a dendritic branch that will allow these quasi-stable receptors to sustain a regenerative depolarization when triggered by an independent gating signal. This process is referred to here as dendritic hold and read (DHR). Because the read-out of the input is not dependent on repetition of the input and information flows in a single-pass manner, DHR can potentially support a feedforward memory architecture.Two contrasting theories have been proposed to explain the mechanistic basis of short term memory. One theory posits that short term memory is represented by persistent neural activity supported by reverberating feedback networks. An alternate, more recent theory posits that short term memory can be supported by feedforward networks. While feedback driven memory can be implemented by well described mechanisms of synaptic plasticity, little is known of possible molecular and cellular mechanisms that can implement feedforward driven memory. Here we report such a mechanism in which the memory trace exists in the form of glutamate-bound but Mg Processing of time-encoded information requires memory. Without some form of short term memory buffer to hold together a temporal sequence, the individual bits of information that are received at any moment in time cannot be properly interpreted. For example, in order to predict the trajectory of a flying ball, it is necessary to hold in mind a temporal sequence of recent positions of the ball. Similarly, the meaning of a sentence can be dramatically different depending on the order of the words and the presence or absence of a single word. The cellular mechanism for such a short term memory buffer is poorly understood. Discussions in the past on the cellular basis of working memory The simplest form of a feedforward memory mechanism is the delay line . It cons2+-blocked state There were two reasons why delay line memory had been dismissed as a memory buffer mechanism in the nervous system. First, plausible biological mechanisms for mediating time delays longer than a few milliseconds had not been identified. The only physiological mechanism considered was the short delay involved with the propagation of an action potential. Delays of a few milliseconds would not provide memory of meaningful duration. Second, without some means to maintain the signal-to-noise ratio, the memory trace would degrade rapidly as the signal was passed from element to element in the linear chain. This is not a problem in the shift register because the flipflop that constitutes the building block for the shift register is a binary device and digital transmission resists signal-to-noise degradation. However, no such analogous feature was known to exist at the level of dendritic integration. Here we propose and demonstrate a new neurophysiological mechanism on dendrites, referred to here as “dendritic hold and read” or DHR, that has the potential to satisfy these two requirements for a biological delay line. We postulate (a) that a memory trace exists in the form of patterns of NMDA receptors in the ligand-bound but MgThe dendritic hold and read (DHR) hypothesis provides testable predictions. Whereas NMDA receptors in the reverberating feedback model are postulated to be in their conducting state 2+ blockade of the NMDA receptors. As a result most of the NMDA receptors would enter into the long-lived, non-conducting, bound-but-blocked state. Indeed, in response to an initial low intensity photolytic release of glutamate, little to no depolarization is observed  in some respects, but unlike classical PPF, which reflects changes in the presynaptic terminal, DHR is exclusively a postsynaptic phenomenon. However, a recent description of a post-synaptic variant of PPF would appear to blur the differences between DHR and PPF Another difference between DHR and PPF is the morphologic substrate for their elementary unit of excitability. For PPF it is individual synapses. For DHR it is functional electrical compartments on the dendritic arbor. Electrical compartmentalization is determined by a combination of factors such as dendritic morphology, active intrinsic conductances, and the pattern of synaptic inputs. Assuming a diffuse pattern of synaptic excitation, the input impedance mismatch at the junction of the thin terminal dendrites to the thick apical trunk, would suggest that the compartments are likely to comprise individual terminal dendrites The most important characteristic of DHR from the perspective of memory architecture is that it does not require the stored information to be repeated identically in order for it to be read-out. Synaptic plasticity mediated mechanisms such as PPF , in contIn summary, these experiments demonstrate that synaptic activity, as simulated with photoreleased glutamate, can leave a transient “memory trace” on individual dendritic branches of pyramidal neurons. This memory trace can persist up to hundreds of milliseconds in the form of bound-but-blocked NMDA receptors and can be conditionally retrieved by an independent gating depolarization. Read-out of the information is in the form of all-or-none local regenerative spikes. DHR represents a new form of information storage and retrieval. We propose that DHR constitutes an elementary building block for feedforward memory.The experiments described here represent the first report of DHR, and accordingly, simply reveals its essential properties. One of the goals was to determine whether the bound-but-blocked NMDA receptors are necessary and sufficient to hold information in DHR. This question could be most clearly demonstrated by minimizing AMPA receptor activation during the priming stimulus. However, it will be important in future studies to demonstrate DHR under conditions of AMPA receptor activation. We know that modest increases in AMPA receptor activation do not prevent potentiation by DHR . Strong A diagram illustrates the essential features of a DHR-based network mechanism . ExperimThe proposed scheme illustrated in The distinctive properties of DHR and the potential power of a feedforward architecture have significant functional implications. Because memory storage in DHR is electrically silent, the amount of energy consumed in holding information would be minimal. Energy is consumed only for the portion of information that is retrieved. This is in sharp contrast to what would be expected for reverberating-activity models of short term memory. Because DHR mediated information storage does not require repetition or forewarning, it is well suited to handle the single pass and arbitrary nature of dynamic stimuli that a nervous system must handle. Together these two properties enable the nervous system to transiently hold massive amounts of information and to perform specific retrieval tasks at later times. It is likely that both feedforward and feedback mechanisms are needed for behavior. Feedback network mediated memory would operate downstream to feedforward memory because DHR can implement two fundamental signal transformations that are needed before the information can be accepted by feedback networks. DHR mediated feedforward memory has the capacity to transform temporal sequences into spatial patterns. DHR also mediates an analogue-to-digital transformation that significantly compresses the size of the input. Individual synaptic events arriving over time are integrated in brief epochs and converted into simple binary outputs. Inputs from hundreds of synapses on a single terminal branch are also integrated as a single electrical compartment. This massive signal compression in time and space may be necessary for processing in the nervous system. It may also explain why memory, even very short term memory, can never have the resolution of a real experience. Feedback networks have their own unique powers such as the ability for plasticity and longer term storage of complex spatially distributed patterns. We speculate that a two stage system, feedback-piggybacked-to-feedforward network, would be a flexible, efficient, and powerful short term mnemonic system.Photolysis of caged glutamate provides the best means for precisely controlling the temporal and spatial pattern of stimulation necessary to test the theory of dendritic hold and read N-[(6-nitrocoumar-7-yl)methyl]-L-glutamic acid) was synthesized in-house. Its preparation is described here. 0.9 g of 7-bromomethyl-6-nitrocoumarin , 1.04 g of H-Glu(OtBu)-OtBu·HCl (3.5 mmol), 1.4 mL of triethylamine (10 mmol) and 6 mL of dry DMSO were added. The mixture was stirred at room temperature for 4.5 h. The reaction mixture was dried under high vacuum, and the residue was purified by column chromatography (5–10% CH3CN/CH2Cl2) to yield N-[(6-nitrocoumar-7-yl)methyl]-L-glutamic acid, di-t-butyl ester as a viscous oil . 1H NMR (CDCl3) 8.22 , 7.77 , 7.72 , 6.54 , 4.14 , 3.15 , 2.38 , 1.98–1.82 , 1.47 , 1.45 . For deprotection, 0.1 g of the di-t-butyl ester was dissolved in 1 mL glacial acetic acid; 1 mL conc. HBr was added, and the reaction mixture was maintained in an ice/water bath for 15 min. The reaction mixture was reduced under vacuum, and the residue was dissolved in 1.2 mL water and purified by reverse-phase HPLC . Product fractions were pooled and lyophilized to yield 0.052 g of N-[(6-nitrocoumar-7-yl)methyl]-L-glutamic acid, or N-Ncm-Glu (68% yield). 1H ((CD3)2SO) 8.49 , 8.15 , 7.71 , 6.63 , 4.05 , 3.17 , 2.32–2.29 , 1.86–1.80 , 1.76–1.66 . Mass spectrum (HR FAB): calculated for C15H15N2O8 (MH+), 351.0828; found 351.0829.Two forms of caged glutamate were used, MNI-Glu and Ncm-Glu. MNI-glutamate was purchased from Tocris or Femtonic. NcmM-Glu  for at least 1 hour prior to recording. Slices were then transferred to a recording chamber and constantly perfused with carbogen-saturated aCSF. Unless otherwise stated, all experiments were performed at temperatures between 32–35°C. Whole-cell recordings were done “blind” using an Axon Instruments Axoclamp 700B Amplifier and pClamp Version 10.2 software was used for data acquisition. Glass recording pipettes (resistance 3–6 megaOhms) were filled with an internal saline solution containing (mM): 135 KCH3SO3, 10 HEPES, 10 NaCl, 1 MgCl2, 0.1 mM K4BAPTA, 2 mM Mg2+-ATP, and 10 mM Phosphocreatine, buffered to pH 7.3 with KOH. Alexa 594 (50–100 µM) was included in the internal solution to allow for visualization of the dendrites. Tetrodotoxin  was included in the aCSF for most experiments. All recordings were done in the presence of the GABAA antagonist bicucuilline methiodide (10–20 µM), GABAB antagonist CGP-35348 (5–20 µM), and 0.1 µM glycine. Caged glutamate  was bath applied. Following a 10 minute baseline, an I-V curve was then established to determine passive properties of the cell. Cells were discarded if Ra increased above 25 mOhms at any point during the experiment. Recordings were done in “current-clamp” configuration and cells were held between −65 to −73 mV. All chemicals and drugs, including ifenprodil and Ro25-6981, were obtained from Sigma-Aldrich.All procedures were approved by the Institutional Animal Care and Use Committee at the University of Maryland School of Medicine. Hippocampal slices were prepared from 2–3 week old Sprague-Dawley rats. Rats were deeply anesthetized and intact hippocampi were quickly removed and placed in chilled artificial cerebral spinal fluid (aCSF) containing (mM): 145 NaCl, 3 KCl, 10 HEPES, 2 CaCl


To prospectively explore the association between non-high-density lipoprotein cholesterol (non-HDLC) and the risks of stroke and its subtypes.A total of 95,916 participants  from a Chinese urban community who were free of myocardial infarction and stroke at baseline time point (2006-2007) were eligible and enrolled in the study. The serum non-HDLC levels of participants were determined by subtracting the high-density lipoprotein cholesterol (HDLC) from total serum cholesterol. The primary outcome was the first occurrence of stroke, which was diagnosed according to the World Health Organization criteria and classified into three subtypes: ischemic stroke, intracerebral hemorrhage, or subarachnoid hemorrhage. The Cox proportional hazards models were used to estimate risk of stroke and its subtypes.th quintile HR=1.33 (1.12-1.59); 5th quintile HR = 1.36 (1.15-1.62); ischemic stroke: 4th quintile HR =1.34 (1.09-1.66); 5th quintile HR = 1.53 (1.24-1.88)).During the four-year follow-up, we identified 1614 stroke events . Statistical analyses showed that hazard ratios (HR)  of serum Non-HDLC level for total and subtypes of stroke were: 1.08 (1.03-1.12) , 1.10 (1.05-1.16) (ischemic), 1.03 (0.96-1.10)  and 0.83 (0.66-1.05) (subarachnoid hemorrhage). HR for non-HDLC refers to the increase per each 20 mg/dl. For total and ischemic stroke, the risks were significantly higher in the fourth and fifth quintiles of non-HDLC concentrations compared to the first quintile after adjusting the confounding factors (total stroke: 4Our data suggest that serum non-HDLC level is an independent risk factor for total and ischemic stroke, and that higher serum non-HDLC concentrations are associated with increased risks for total stroke and ischemic stroke, but not for intracerebral and subarachnoid hemorrhage. Stroke is a major healthcare problem and a serious economic burden to society. In China, stroke is the leading cause of death and long-term disability. Currently, more than 7,000,000 individuals suffer from stroke, and there are 2,000,000 individuals with newly diagnosed stroke each year ,2.Atherosclerosis and lipoprotein disorders have been indicated as critical pathological changes during the development of stroke . Non-higThe Kailuan study was a prospective cohort based in Kailuan community in Tangshan city, which is a large and littoral modern city located in the southeast of Beijing. The detail information about Kailuan Study has been described previously –14. FromBlood samples were collected from an antecubital vein of participants in the next morning after overnight fast (>8 hours) and then transferred into vacuum tubes containing EDTA (Ethylene Diamine Tetraacetic Acid). All the blood samples were processed and analyzed using an auto-analyzer  at the central laboratory of the Kailuan General Hospital.Level of total cholesterol (TC) in serum was measured using endpoint test method. HDLC and LDLC were measured with direct test method. Triglyceride (TG) was measured by GPO method . Non-HDLC level was determined by subtracting serum HDLC from serum TC.Information on demographic variables  was collected via questionnaires. The education level was categorized as “college or higher”, “middle school”, or “illiteracy or primary school”.2. BMI was classified as ideal if it was <25 kg/m2. Systolic Blood pressure (SBP) and Diastolic Blood pressure (DBP) were measured twice in the seated position using a mercury sphygmomanometer. The average of two readings was used in the analyses.Information on smoking and physical activity was also collected via questionnaires. Weight and height were measured during the interview and body mass index (BMI) was calculated as weight (kg)/height (m)The participants were followed up by face-to-face interviews at every two-year routine medical examination until December 31, 2010 or to the event of interest or death. The follow-ups were performed by hospital physicians, research physicians, and research nurses who were blinded to the baseline data. The outcome information for the participants without face-to-face follow-up was obtained by referring to death certificates from provincial vital statistics offices, discharge summaries from the 11 hospitals, and medical records from medical insurance.The primary outcome was the first occurrence of stroke; either the first nonfatal stroke event or a stroke death. A nonfatal stroke was defined as a focal neurological deficit of sudden onset and vascular mechanism that lasted >24 hours. Stroke was diagnosed according to the World Health Organization (WHO) criteria  combinedThe participants were classified into 5 groups according to quintiles of serum non-HDLC level. In addition, medians and proportions of potential risk factors for stroke among these 5 groups were calculated. Continuous variables between groups were compared by Variance analysis, while categorical variables were analyzed by Chi-Square test. The raw and adjusted hazard ratios (HRs) with 95% confidence intervals (CIs) were calculated from the Cox proportional hazards model. There were 11 hospitals participating in the current study. Thus, the cox proportional hazard model with a sandwich covariance matrix as a random effect was used to account for the potential effects due to different treatment centers. Other potential confounders included age, gender, BMI, hypertension, diabetes, smoking and drinking status , physical activity status , HDLC, and triglyceride. A trend test was used to examine the relationship between different quartile levels of serum non-HDLC and the risk of stroke. Outcome assessors and statisticians were independent and were blinded to the participants’ baseline information. All statistical analyses were performed with the SAS software, version 9.2 . All CIs were estimated at the 95% level and signiﬁcance was set at a P value of <0.05 (2-sided).In our study, all participants were followed up to the event of interest or death through multiple ways. For participants who took the every two-year routine medical examination, they were followed up by face-to-face interviews by hospital physicians and nurses. For those without face-to-face interviews, their follow up information was obtained by checking death certificates from provincial vital statistics offices, discharge summaries from the 11 designated hospitals, or medical records from medical insurance companies. However, we were did not collect the information in this study regarding the proportion of the cohort followed up by face-to-face or by records only.During the four-year follow-up, we identified a total of 1,614 stroke cases, including 1,156 ischemic, 416 intracerebral hemorrhage, and 42 subarachnoid hemorrhage. Multivariate analysis in As demonstrated in In order to determine the effect of potential confounding factors on the significant association between serum non-HDLC level and stroke incidences shown in The results of the present large prospective study, based on a community population in China with greater than 96,000 participants, suggested that serum non-HDLC level is an independent risk factor for stroke and one of its subtypes, ischemic stroke. Our data identified a positive association between serum non-HDLC level and the incidences of total stroke and ischemic stroke.Although the positive association of serum non-HDLC level with the risk of coronary heart diseases has been well documented ,16,17,, We also analyzed the relationship between the serum non-HDLC level and occurrence of hemorrhage stroke subtype. Our study reported a higher proportion of hemorrhagic stroke than those reported in Western countries, which is consistent with some previous studies conducted in China where the proportion of hemorrhagic strokes was reported to be 17.1% to 55.4% ,27, highCompared to previous reports, our study demonstrated unique strengths and advantages. First, our data were generated from a prospective cohort study with a very large sample size . Of importance, the Chinese adult participants enrolled in our study showed a similar baseline participants’ distribution as other epidemiological studies in China ,33. AddiThe present large prospective cohort study based on a Chinese community population showed that the serum non-HDLC is an impendent risk factor for stroke and its certain subtypes, and that high serum non-HDLC concentration is associated with the increased risk for total stroke and ischemic stroke, but not for intracerebral hemorrhage and subarachnoid hemorrhage. As a non-expensive and easily tested biomarker, non-HDLC may represent an important alternative screening marker for primary prevention of ischemic stroke among Chinese adults.


An additional group of rats was tested following adrenalectomy. All FDR rats showed a statistically significant increase in heroin seeking compared to the sated rats. No statistically significant effects for treatment with α-helical CRF, R121919, RU486 or adrenalectomy were observed. These findings suggest that stress may not be a critical factor in the augmentation of heroin seeking in food-restricted rats.Drug addiction is a chronic disease characterized by recurring episodes of abstinence and relapse. The precise mechanisms underlying this pattern are yet to be elucidated, but stress is thought to be a major factor in relapse. Recently, we reported that rats under withdrawal and exposed to a mild chronic stressor, prolonged food restriction, show increased heroin seeking compared to sated controls. Previous studies demonstrated a critical role for corticotropin-releasing factor (CRF) and corticosterone, hormones involved in the stress response, in acute food deprivation-induced reinstatement of extinguished drug seeking. However, the role of CRF and corticosterone in chronic food restriction-induced augmentation of drug seeking remains unknown. Here, male Long-Evans rats were trained to self-administer heroin for 10 days in operant conditioning chambers. Rats were then removed from the training chambers, and subjected to 14 days of unrestricted (sated rats) or a mildly restricted (FDR rats) access to food, which maintained their body weight (BW) at 90% of their baseline weight. On day 14, different groups of rats were administered a selective CRF Stress is consistently reported by drug users as a factor in subjective craving as well as in the initiation, maintenance, and relapse of drug use  decrease BW and augment drug seeking in rodents, they can have quite different metabolic and behavioral effects on the organism. For example, Fulton et al.  demonstrAlthough the extrahypothalamic CRF system is critically involved in acute FD-induced reinstatement of heroin seeking  were used. Before surgery, animals were pair-housed for 1 week in the animal care facility (ACF) under reverse light/dark conditions (lights OFF at 0930). Following intravenous (IV) catheterization, and 2 days of recovery, rats were single-housed in plastic shoebox cages before being transferred to operant conditioning chambers for drug self-administration. Following self-administration training, rats were returned to the ACF and single-housed in shoebox cages for the drug withdrawal phase. Except for the withdrawal phase, all rats were given unrestricted access to food and water. Rats were treated according to the Canadian Council on Animal Care guidelines, and approval was granted by the Concordia University Animal Research Ethics Committee.Rats were implanted with IV silastic catheters  under xylazine/ketamine . Three centimeters of silastic catheter was inserted through a small incision on the right jugular vein, and secured using silk sutures. The remainder of the catheter was passed subcutaneously to the skull, attached to a modified 22-gauge cannula  and anchored to the skull using dental cement and 5 jeweler's screws. Some rats (Experiment 1A) were also implanted with a 22-gauge guide cannula (Plastics One) aimed 2 mm above the right or left lateral ventricle  to allow for i.c.v. injections.Following surgery, rats were administered buprenorphine  and penicillin  to reduce pain and prevent infection. Catheters were flushed daily with heparin/gentamicin (7.5 IU +12.0 mg/rat) to prevent blockage and infection.Bilateral adrenalectomy (ADX) surgeries were performed on withdrawal day 10 on rats in Experiment 2B, under isoflurane USP anesthetic. The adrenal glands were rapidly removed via the dorsal approach, and the rats were given ketoprofen injections  following surgery to reduce pain. Sham-operated rats were exposed to the same procedure as the ADX rats, with the exception that the adrenal glands were not removed. After surgery, the ADX rats were given physiological saline (0.9% NaCl) in their drinking bottles.Rats were housed individually in operant conditioning chambers  enclosed in sound attenuating wooden compartments equipped with a fan. Each chamber was fitted with two retractable levers (Coulbourn Instruments) mounted 9 cm above the floor of the right sidewall. Responses on the “active” lever activated the infusion pump and a cue-light/tone  located above the lever. Responses on the “inactive” lever had no programmable consequences. Rats were attached to the infusion pump via a liquid swivel  and Tygon tubing shielded with a metal spring.Heroin HCl  was dissolved in sterile saline (5.0 mg/ml) and then further diluted with saline, for each rat according to BW to yield 0.1 mg/kg/infusion.1 receptor antagonist, was kindly supplied by Dr. Kenner Rice . R121919 was dissolved in a 20% β-Cyclodextrin (Sigma-Aldrich) sterile saline solution to a concentration of 10.0 mg/ml and adjusted to a pH of 4.5. The antagonist was injected (s.c.) at a final dose of 20.0 mg/kg. A solution of 20% β-Cyclodextrin mixed in sterile saline solution was used as a vehicle injected at a volume of 2.0 ml/kg. A similar dose of R121919 reduced both drug self-administration and anxiety-like behaviors , was dissolved in sterile water to a concentration of 5.0 μg/μl or 12.5 μg/μl. α-helical CRF was injected i.c.v. over 2 min at a rate of 1.0 μl/min for a final dose of 10.0 or 25.0 μg/rat. Sterile water was used as a vehicle. Injections were made using a syringe pump  connected to a 10 μl Hamilton syringe. This syringe was attached via polyethylene-20 tubing to a 28-gauge injector (Plastics One) that extended 2 mm below the guide cannulae. The injector was kept in place for 1 min after the injections to allow for proper diffusion of the drug. The doses for α-helical CRF were based on previous reports , 1% Tween-80 (Sigma-Aldrich), 2–3 drops of 1N HCl, and sterile water mixture which also served as the vehicle. RU486 and vehicle solutions were adjusted to a pH of ~5.6 and injected (i.p.) at a dose of 30 mg/kg (RU486), or a volume of 1 ml/kg (vehicle). A similar dose was shown to attenuate stress-induced reinstatement of alcohol seeking , resulted in activation of the drug pump  and the initiation of a 20 s timeout during which the house-light was turned off and the cue light/tone complex above the active lever was activated. During the timeout period, active lever responses were recorded but not reinforced. Following each 3-h session, the active lever was retracted whereas the inactive lever was not retracted until 1 h before the first session of the following day. Inactive lever responses were recorded but had no programmable consequences.Following self-administration training, rats were individually housed in the ACF, and given unrestricted access to food and water for one drug-washout day. Rats were then divided into two groups: food restricted (FDR) or Sated that were matched according to BW, number of infusions, and active lever responses across the last 5 days of training. Following the washout day, FDR rats had their food removed and were fed ~15 g of rat chow at 1330. The amount of food was adjusted through 14 days of food restriction to maintain the food restricted rats' BW at ~75–80% of the Sated rats and 90% of their baseline BW.Rats were treated as in Experiment 2A, except for the ADX surgery that was performed on day 10 of withdrawal. Following surgery, rats were given 25 g of food and allowed 1 day of recovery, after which they were re-restricted to the previously described regimen and allowed 2 extra days of withdrawal to ensure their BWs reached criteria.On withdrawal day 14, rats were returned to the operant conditioning chambers and attached to the metal spring. The drug-seeking test consisted of a 1-h session during which active lever responses had the same consequences as in training excluding the availability of the drug. A subcutaneous injection of R121919  was administered 30 min before the test session.The testing procedure was similar to the one described for Experiment 1A, except that the rats were given an ICV injection of α-helical CRF  10 min before the test session.This experiment was designed to verify the efficiency of the α-helical CRF treatment used in Experiment 1B. Previously, α-helical CRF has been shown to have anxiolytic properties  or vehicle in the ACF 45 min before the test session.Testing procedure was similar to the one described for Experiment 1A, except that, other then the ADX surgery, no further treatment was given before the test.Immediately following the drug-seeking test (1030), tail blood was collected, and plasma was separated by centrifugation at 10,000 rpm for 10 min. Samples were stored at −80°C. Plasma samples were analyzed for corticosterone levels using a corticosterone specific enzyme-linked immunosorbent assay (ELISA) kit . The reported detection sensitivity for the kit is 27.0 pg/ml.All analyses were conducted using SPSS software . Training data for all rats were analyzed using a repeated measures ANOVA, with training day (1–10) as a within-subjects factor and the number of active lever responses, inactive lever responses or number of infusions as the dependent variables.Number of responses on the active and inactive levers during the drug-seeking test sessions were analyzed separately using Two-Way ANOVAs. Antagonist dose  and feeding condition  served as between subject factors.t-tests. Latency to consumption and number of approaches were the dependent variables.The effect of treatment with α-helical CRF  on the open field behavior was analyzed using two independent samples Number of responses on the active and inactive levers during the test session were analyzed separately using Two-Way ANOVAs. Antagonist dose  or surgery condition  and feeding condition  served as the between subject factors.Plasma corticosterone levels (ng/ml), sampled following the test sessions in Experiment 2, were analyzed using a Two-Way ANOVA. Antagonist dose  or surgery condition  and feeding condition  served as the between subject factors.post-hoc tests and the critical threshold for statistically significant results was set at p ≤ 0.05.In all analyses, statistically significant interactions were investigated with the appropriate All rats acquired reliable heroin self-administration behavior. Mean ± SEM number of infusions and number of active and inactive lever responses made on the last day of heroin self-administration training, for each experiment, are shown in Table n = 5), FDR-20.0 (n = 7), Sated-0.0 (n = 6), Sated-20.0 (n = 7). On the test day, average BW of the rats in the Sated group  was statistically significantly greater than that of the rats in the FDR group  = −10.12, p < 0.001).Five rats were removed due to catheter leakage, failure to train, or detached head-cap. Thus, the final analysis included 25 rats in 4 experimental groups: FDR-0.0  = 6.91, p = 0.016, η2 = 0.24; Figure The mean number of active lever responses performed by the rats in the FDR group was almost three times higher than the lever responses made by the Sated group during the drug-seeking test. The robust effect was confirmed by a statistically significant main effect of feeding condition . No statistically significant effects were found for either the antagonist dose or for the feeding condition × antagonist dose interaction. No significant effects were observed for inactive lever responding on test day  = 2.80, p = 0.038; Figure t(5) = 3.13, p = 0.021; Figure Rats in the α-helical CRF-treated group demonstrated a statistically significant decrease in latency to first food consumption and the number of approaches prior to first consumption, compared to vehicle controls  during the drug-seeking test. No statistically significant effects for antagonist dose or feeding condition × antagonist dose interaction were observed. The FDR group also responded more on the inactive lever than the Sated group  and antagonist dose , and a significant feeding condition × antagonist dose interaction effect . Although this significant interaction effect was clearly driven by the dramatic increase in corticosterone levels induced by the RU486 treatment in the FDR rats, corticosterone levels in this group were not statistically significantly different from the sated, RU486-treated rats , probably due to the sample size in the FDR, RU486-treated group (n = 3). Further post-hoc tests are described in Figure Some corticosterone samples that were collected immediately following the test session were not included in the final analysis due to an unacceptable variability between the duplicates in the ELISA kit (>30%). Administration of RU486 resulted in increased levels of corticosterone in both the sated and FDR rats. However, treatment with RU486 resulted in considerably higher corticosterone levels in the FDR group Figure . ANOVA rF1, 1 = 12.80,Three rats were removed due to catheter leakage, failure to train or detached head-caps. Thus, the final analysis included 29 rats in 4 experimental groups: FDR-Sham = 6, FDR-ADX = 7, Sated-Sham = 7, Sated-ADX = 9.F = 108.85, p < 0.0001] than the FDR group's BW . No statistically significant effects on BW for surgery group or the feeding condition × surgery group interaction were observed.On test day, the average BW of the Sated group  was statistically significantly greater  during the drug-seeking test. No statistically significant effects for surgery group or the feeding condition × surgery group interaction were observed. No significant effects were observed for inactive lever responding on test day , and were not affected by food restriction (data not shown).1-R antagonist, or α-helical CRF, a non-specific CRF-R antagonist, did not result in a statistically significant reduction in heroin seeking in food restricted rats under withdrawal. Similarly, neither treatment with RU486, a glucocorticoid receptor antagonist, nor adrenalectomy affected heroin seeking in this model.Recent work in our laboratory has demonstrated an augmentation of heroin seeking in chronically food restricted rats, under withdrawal  are observed following acute, but not chronic stress. In contrast, chronic stress results in lowered levels of CRF1-R and c-fos mRNA in the PVN  tissue levels in the nucleus accumbens (NAc), and reduced levels in the prefrontal cortex (PFC), were found 1 week following the completion of a chronic treatment (13 days) with CRF  lever on the test day was observed in the α-helical CRF-treated sated group. Recently, CRF-R antagonism was shown to reduce cue-induced reinstatement of drug seeking  remains unaffected, in spite of a blunted HPA axis response and lowered concentrations of corticosterone  was greater than that in the sated group (~74%) following RU486 treatment. We speculate that these differences in magnitude can be explained by the lack of negative feedback from circulating corticosterone, which resulted in amplification of the increased corticosterone levels that are typically found in food restricted rats.There is evidence that the augmentation of drug seeking following food restriction can be modulated by homeostatic mechanisms that are triggered by the hunger state (Cabeza de Vaca and Carr, An additional peripheral signal that is involved in energy balance is leptin, an anorexigenic hormone that is secreted by peripheral adipocytes (Friedman and Halaas, In conclusion, we suggest that pathways involved in the acute stress response are not critical for the expression of augmented drug seeking in food-restricted rats under withdrawal. Our findings, however, do not exclude a role for food restriction-induced prolonged increases in CRF and corticosterone in the induction of downstream brain adaptations, which in turn may drive the augmented drug seeking observed in rats under withdrawal from heroin.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Thrombophilia represents a risk factor both for idiopathic and secondary osteonecrosis (ON). We evaluated whether clotting changes in idiopathic ON were different from corticosteroid-associated ON. As platelet-rich plasma has been proposed as an adjuvant in surgery, we also assessed whether platelet and serum growth factors were similar to those in healthy subjects.β1, platelet-derived growth factor-BB (PDGF-BB), and vascular endothelial growth factor were assayed in the supernatants of thrombin-activated platelets, in platelet lysates, and in serum from 14 ON patients and 10 controls. 18 patients with idiopathic ON and 18 with corticosteroid-associated ON were compared with 44 controls for acquired and inherited thrombophilia. Platelet factor 4 (PF4), transforming growth factor- Idiopathic ON patients had higher plasminogen levels (median 118%) than controls (101%) (p = 0.02). Those with corticosteroid-associated ON had significantly higher D-dimer (333 ng/mL) and lower protein C levels (129%) than controls . The frequency of inherited thrombophilia was not different from the controls. No statistically significant differences were found between idiopathic and corticosteroid-associated ON. 20 of the 36 ON patients were smokers. (The controls were selected from smokers because nicotine favors hypercoagulability). ON patients had significantly higher serum PF4 levels  and PDGF-BB levels (3.1 ng/mL) than controls . Acquired hypercoagulability was common in both ON types, but the specific changes varied. The release of GF from platelets was not affected, providing a biological basis for platelet-rich plasma being used as an adjuvant in surgical treatment. Osteonecrosis (ON) of the femoral head may lead to joint collapse and arthritis. In the late stages of the disease, when pain, stiffness, and disability cannot be controlled by non-surgical means, the treatment is total hip replacement. At the early stages, when less invasive treatments to preserve the femoral head such as core decompression, osteotomy, or vascularized bone grafting—or even non-invasive therapy, such as pharmacological measures, electrical stimulation, shock waves, and electromagnetic fields —can prevIt is commonly postulated that ON is caused by reduced blood flow to the bone . VasculaOn the basis of the high concentration of growth factors (GFs) released by α-granules, platelet-rich plasma (PRP) has been proposed as an adjuvant to improve angiogenesis and osteogenesis in the surgical treatment of ON . HoweverWe evaluated whether patients affected by idiopathic ON had different plasmatic and genetic abnormalities of thrombophilia and fibrinolysis from patients with corticosteroid-associated ON. We also assessed whether the platelet levels of osteogenic and angiogenic GFs were similar to those of healthy subjects.5/μL were excluded.The study protocol was approved by the institutional ethical committee on human research and was performed according to the Helsinki Declaration of 1975, as revised in 2000. The study included patients with idiopathic and corticosteroid-associated ON of the femoral head who had been diagnosed by clinical and radiological criteria and were hospitalized for hip surgery. Before surgery, an informed consent document was signed, the familial and clinical history was registered, and a sample of blood was taken. The patients had not taken heparin, oral anticoagulants, aspirin, or other platelet antiaggregants in the 2 weeks preceding blood collection. Patients with platelet disorders or platelet number less than 1 × 1018 patients had idiopathic ON and in 18 individuals the disease developed after corticosteroid treatment. Contrary to other studies, we did not find any case of alcohol-related ON. The consumption of alcohol was occasional or limited to half or one glass of wine per meal. There were no statistically significant differences in age and sex between idiopathic and corticosteroid-associated ON. The patients were compared with 44 healthy individuals who were similar to the patients regarding age and sex, and who had no clinical disorder or history of thrombosis . A familIn a group of 14 ON patients (mean age 35 (19–64) years, 13 males) selected on the basis of surgery, platelet factor 4 (PF4) and GF levels in platelets and serum were compared with those in 10 healthy controls. 12 patients were affected by idiopathic ON and 2 patients by secondary ON (1 by posttraumatic ON and 1 by corticosteroid-associated ON). Age and sex were similar in the controls and patients.g for 10 min. The plasma was frozen in aliquots at –70°C. The samples for the assays of PF4 and GF were centrifuged at 180 g for 5 min in order to separate PRP from erythrocytes and leukocytes. Then the PRP was transferred to a clean tube and centrifuged at 600 g for 15 min. The platelets were pelleted at the bottom of the tube; platelet-poor plasma (PPP) in the upper phase was carefully transferred to a clean tube. Platelet number was adjusted to 1 × 106/μL with PPP. Then the PRP was divided into 2 aliquots. In the first aliquot, platelets were lysed by 2 cycles of freeze-thawing at –80°C. In the second aliquot, the platelet release reaction was induced by adding 5 U/mL bovine thrombin  for 30 min at room temperature, the samples were centrifuged, and the supernatants (from platelet release) were stored at –80°C prior to assay. PF4 and GF were also determined in serum, which was stored at –80°C after centrifugation at 600 g for 45 min. Blood for genetic analysis was drawn in tubes containing ethylenediaminetetraacetic acid and stored at –20°C.For the clotting tests and for PF4 and GF assays, the blood was collected in 0.129 M sodium citrate. Within 2 h of collection, the samples for the clotting tests were centrifuged at 600 Antithrombin III was determined with a chromogenic assay . The percentage of antithrombin III activity in the samples was extrapolated from a standard curve of scalar dilutions of Calibration Plasma (Instrumentation Laboratory) according to the linear regression function.Protein C activity was determined with a clotting test  based on the prolongation of APTT in the presence of activated protein C, generated by an activator derived from snake venom. The percentage of protein C activity was extrapolated by a standard curve of scalar dilutions of Calibration Plasma according to the linear regression function.D-dimer was measured by enzyme immunoassay . The standard curve was plotted using the point-to-point method.Plasminogen was determined with a chromogenic assay . The percentage of activity of plasminogen in the samples was extrapolated from a standard curve of scalar dilutions of Calibration Plasma according to the linear regression function.PAI-1 was detected in citrated plasma by enzyme immunoassay (AssayMax Human Plasminogen Activator Inhibitor-1 (PAI-1) ELISA Kit; Assaypro, St. Charles, MO). The standard curve was plotted by regression analysis using log-log curve fit.max ImmunoAssay System: Promega Corp., Madison, WI; Quantikine Human PDGF-BB: R&D Systems Inc., Minneapolis, MN; DuoSet Human VEGF: R&D Systems). Before assay of TGF-β1, the samples were activated with 1N HCl to pH 3.0 or lower for 15 min and then neutralized with 1N NaOH to pH 7.6.PF4, transforming growth factor-β1 (TGF-β1), platelet-derived growth factor-BB (PDGF-BB), and vascular endothelial growth factor-A (VEGF-A) were determined in platelet releasates, lysates, and in serum with enzyme immunoassays according to the directions of the manufacturers  and stored at –20°C.The single point mutation (G to A at position 1691) of the factor V gene (factor V Leiden) was detected using real-time PCR  on the LightCycler 1.2 instrument (Roche Applied Science). The amplification program consisted of a first denaturation at 95°C for 30 seconds and 45 cycles of amplification .The prothrombin gene mutation 20210A was detected using real-time PCR (Factor II (Prothrombin) G20210A Kit; Roche Applied Science) on the LightCycler 1.2 instrument. The amplification program consisted of a first denaturation at 95°C for 30 seconds and 45 cycles of amplification .The PAI-1 gene 4G/5G polymorphism was detected using real-time PCR  on the LightCycler 1.2 instrument. Five μL of the DNA template was added to a master mix composed of 2.2 μL 4 mM MgCl2, 2 μL LC DNA Master Hybridization Probes and 1 μL of each of the primers huPAI 4/5G, in a total volume of 20 μL. The amplification program consisted of a denaturation step at 95°C for 60 seconds followed by 37 cycles of amplification .http://www.quantitativeskills.com/sisa/). Quantitative results are reported as arithmetic mean, standard deviation (SD), median, and minimum-maximum range. Because normal distribution and homogeneity of variance were not verified by Levene's test, the differences among the groups were evaluated with Kruskal-Wallis test, and the differences between ON groups, or each ON group and the negative control, were evaluated with the Mann-Whitney U-test. The differences between the concentrations of GFs in serum and in platelet releasates or lysates were evaluated with the Wilcoxon signed rank test. The differences were considered significant at p-values < 0.05.Statistical evaluations were performed using StatView for Windows version 5.0.1 . The statistical power was calculated with Simple Interactive Statistical Analysis (SISA) , and they were increased in ON (Mann-Whitney U test: p = 0.006). In particular, smokers with idiopathic ON showed statistically significantly higher D-dimer levels than controls. In these patients, a trend was also evident regarding higher plasma PAI-1 levels than controls . No statA high degree of variability was seen in platelet and serum levels of PF4 and GFs, both in patients and in healthy controls. The levels of GFs in releasates were higher than in serum; in particular, PDGF-BB was 2.7- (2.1-) fold higher, TGF-β1 was 1.7- (2.3-) fold higher and VEGF was 1.5- (0.3-) fold higher. The lysate:serum ratio was higher than the releasate:serum ratio: for PDGF-BB it was 3.4- (1.8-) fold higher, for TGF-β1 it was 4.8- (4.5-) fold higher and for VEGF it was 1.3- (0.5-) fold higher. Even though the GF levels in releasates and lysates from ON patients were higher than those from healthy controls, no statistically significant differences were found. Instead, the serum concentrations of PF4 and PDGF-BB in ON patients were statistically significantly higher than in the controls .Homozygosity or heterozygosity for factor V Leiden was present in only 2 patients affected by corticosteroid-associated ON. Factor V Leiden was not detected in any patients with idiopathic ON or in any controls. Homozygosity for the prothrombin gene mutation was not found in any patients or controls; heterozygosity was present in only 2 patients with idiopathic ON and in 1 control. The genotypes 4G/5G and 4G/4G of the PAI-1 gene were demonstrated in 7 and 6 of 18 patients with idiopathic ON, and in 9 and 4 of 18 patients with corticosteroid-associated ON. The frequency of 4G/4G homozygosity was also high in healthy controls (19/44), while 16 of the 44 control subjects were heterozygous.We hypothesized that the alterations in the clotting system or in fibrinolysis in idiopathic ON may be different from those in corticosteroid-associated ON. The secondary hypothesis was that the levels of GFs in ON platelets are similar to or higher than in those of healthy subjects, thus supporting the use of PRP as an adjuvant for the treatment of ON.Although the idiopathic and corticosteroid-associated ON groups were homogenous regarding age, sex, and stage, one limitation of the study may be the low number of cases. The 2 types of ON showed different alterations in the clotting tests. In particular, higher levels of plasminogen were detected in idiopathic ON while lower protein C activity and higher concentrations of D-dimer were found in corticosteroid-associated ON.The high plasminogen levels in idiopathic ON may have several explanations. Some degree of synovial inflammation has been shown in ON and it was hypothesized to play a role in the disease progression . PlasminNormal or low levels of plasminogen have been reported by other authors in both ON and Perthes' disease, which has a similar pathogenesis. For all the parameters examined, no statistically significant differences between idiopathic and corticosteroid-associated ON were found. Corticosteroid-associated ON showed peculiar alterations, consisting of a decrease in protein C and in an increase in D-dimer relative to healthy controls. A possible limitation of the finding of a reduction in protein C in corticosteroid-associated ON was the fact that the statistical power was 72% for α = 0.05; thus, our findings require to be confirmed by further studies. Other authors have found reduced protein C activity in non-traumatic ON . In PertInstead, the statistical power analysis based on difference between corticosteroid-associated ON and controls regarding D-dimer levels revealed a power of 95% for α = 0.01. An increase in D-dimer was reported in 25% and 21% of patients with idiopathic or secondary ON, respectively, as a consequence of the hypercoagulability that also induced ON . CorticoWe found a low frequency of inherited thrombophilia both in idiopathic ON and corticosteroid-associated ON. The frequencies of factor V Leiden and prothrombin gene mutations were similar to those in the controls, contrary to what has been asserted by some authors who have hypothesized that idiopathic ON might be favored by factor V Leiden or prothrombin gene mutation .http://www.salute.gov.it/imgs/C_17_pubblicazioni_1161_ulteriorallegiati_ulterioreallegato_0_alleg.pdf). Other researchers have found a strong association between tobacco consumption and risk of ON. Interestingly, 55% of the ON patients were smokers, as compared to a rate of 23% in the general population (Activities for the prevention of smoking—2009 report, Ministry of Health, Italy, In our study, D-dimer levels were higher in smokers with ON than in smokers in the control group. This result was more evident in smokers with idiopathic ON. In these patients, a trend of higher plasma levels of PAI-1 was also found. Thus, we could hypothesize that smoking is only one of several risk factors for idiopathic ON. Other causes may induce hypercoagulability, which is important for ON pathogenesis. In smokers with corticosteroid-associated ON, D-dimer levels were not statistically significantly different from those in the controls, which is contrary to what was observed when we considered smokers and non-smokers together. In this group, other causes, independent of smoking and present also in non-smokers, could have induced the increase in D-dimer. In order to establish a scientific basis for the use of platelet-rich plasma in ON, we determined the levels of some osteogenic and angiogenic GFs in platelet lysates and releasates. The levels of platelet GFs are known in healthy blood donors , but theIn conclusion, we did not detect statistically significant differences in clotting tests between idiopathic and corticosteroid-associated ON. In comparison with healthy controls, idiopathic ON showed higher plasminogen activity; corticosteroid-associated ON had lower protein C levels and higher D-dimer levels. However, these results require to be confirmed by further studies on a larger number of patients, particularly the data regarding plasminogen and protein C. In both groups of patients, the frequency of heritable thrombophilia and fibrinolysis was similar to that for the controls. Thus, the results of this preliminary study support the idea that both idiopathic and corticosteroid-associated ON are favored by acquired hypercoagulability, but at a subclinical level. The high levels of PF4 in releasates showed that platelet release reaction is not affected in ON. The concentrations of GFs in releasates and lysates from ON patients were similar to those in healthy subjects. This result provides a biological basis for the use of PRP in ON treatment.


The cytotoxicity of alpha- and beta-asarone was investigated with the BrdU assay in HepG2-cells. Alpha-asarone was found to be more toxic than beta-asarone after 24 hours of treatment. Investigation of the genotoxicity using the micronucleus assay in the HepG2-cell system showed that only after metabolic activation by a liver microsomal preparation, beta-asarone was able to induce micronuclei at concentrations higher than 50 μg/mL. Established cell lines can be used to determine cytotoxicity, but for the investigation of genotoxicity, theirapplication is rather limited.Studies of the genotoxicity of phytoherbal preparations or isolated natural compounds are needed to in order to demonstrate their safety, and are prerequisites for approval procedures. For these studies, short-term N-demethylase, catalase, peroxidase, NAD(P)H:cytochrome c reductase, cytochrome P450 reductase, and NAD(P)H, Quinone oxidoreductase and phase II enzymes such as epoxide hydrolase, sulfotransferase, glutathione S-transferase (GST), uridine glucuronosyl transferase, and N-acetyl transferase. It has been demonstrated that cultivated HepG2 cells contain mixed-function oxidases [in vivo. This problem can be solved using an exogenous activation mixture containing mammalian microsomal enzymes [Cell lines of liver origin are widely used in biomedical research involving xenobiotic metabolism including genotoxicity studies. The HepG2 cell line is one of the most often used cell systems in xenobiotic and metabolism research, which is biochemically and physiologically characterized in detail. This cell line is also qualified for the detection of environmental and dietary genotoxicants . This ce enzymes . The mosBased on new regulatory requirements for pharmaceutical preparations made from plants of the genus Asarum, which also contain aristolochic acids , it woulIn HepG2-cells, alpha- and beta-asarone show a different cytotoxicity in the BrdU-assay without metabolic activation, as demonstrated in in vitro results on previously used drugs containing asarones requires the absence of beta-asarone. For this purpose, an analytical method is necessary which separates both isomers of asarone and detects low concentrations of the compounds. The values for the daily intake of alpha-asarone containing preparations should be regulated according to the TTC concept [After compiling the results, both enantiomers of asarone show cytotoxicity, but only beta-asarone induces genotoxicity. On the other hand, alpha-asarone is more cytotoxic. For pharmaceutical preparations based on plants from the genus Asarum, which also contain essential oils with asarones, there is obviously no genotoxic risk based on the content of alpha-asarone, but the cytotoxicity of this compound should be taken into account. The transfer of these  concept , i.e. thHepG2 cells were obtained from DSMZ Braunschweig, Germany, the cell culture media and the fetal calf serum (FCS) were supplied from Biochrom AG, Berlin.Alpha- and beta-asarone and all other chemicals were purchased from Sigma-Aldrich, Steinheim. The Cell Proliferation ELISA-kit (BrdU colorimetric) was obtained from Roche Diagnostics GmbH, Mannheim, the S9-mix  from TRINOVA, Gießen. The plasticware for cell cultivation was supplied by Greiner bio-one, Frickenhausen.2 flasks in RPMI 1640, supplemented with 10% FCS  for 3 or 4 days before the experiments were started. Only passages from number 4 to 20 were used for the experiments. The cells were counted for the inoculation by Casy Cell Counter . For detachment of the cells, EDTA-Trypsin-solution (0.02/0.05% in PBS) was used.The cells were cultivated in 75 cmFor determination of the cytotoxicity, cell proliferation was measured using Cell Proliferation ELISA, BrdU (colorimetric) . HepG2 cells were seeded into 96-well plates at the concentration 20 000 cells/well and cultivated at 37°C, 5 % CO2 for 24 h. Subsequently, the cells were exposed to medium containing the asarones. After 24 h, 20 μL BrdU (100 μM) per well were added and incubated at 37°C for 2 h. The following procedures were carried out according to the manufacturer’s instructions. The changes in absorbance were recorded by a photometer  at 450 nm wavelength. The DNA synthesis was calculated in comparison to the controls (with solvents) and shown in relation to the used concentration of alpha- or beta-asarone .2) 37 500 cells were seeded in 2.5 mL RPMI medium + 10% FCS for 24 h. Appropriate dilutions of asarones (dissolved in DMSO) and DMSO (controls) were added to the cells directly. The cells were then cultivated for 24 h. When the S9-mix was used, the medium was changed and 625μL S9-mix per flask suspended in 1.875 mL medium was added for 6 h. After that, the medium was removed and fresh RPMI medium plus 3 μg/mL Cytochalasin B was added. After 24 h, the cells were detached from the flask bottom with EDTA-Trypsin-solution, diluted with medium, centrifuged  and the pellet was suspended in fresh medium. After counting the cells (Casy), a cell suspension with 2×105 cells/mL fresh medium was prepared. 300 μL of the cell suspension were now centrifuged on a slide . For each culture flask two slides were prepared. After fixation in 90% methanol (−20°C) the slides were stained two times for 15 sec with a solution of acridine orange (0.0125% in PBS) and washed with PBS for 10 min. For determination of the number of micronuclei per 1 000 binuclear cells, the slides were counted by fluorescence microscopy .The determination of the micronucleus formation was performed according to a published protocol . BrieflyData in tables and figures were presented as the mean ± SD. Differences between groups were assessed by the Mann-Whitney U test. A probability of p < 0.05 was considered significantly different. The test of normally distributed data was performed by the Shapiro-Wilk test. All tests were performed using the Statgraphics Plus 5.1 program.